ELISA - General Tips and Troubleshooting

Enzyme-linked Immunosorbent Assays (ELISAs or EIAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme (e.g. alkaline phosphatase, horseradish peroxidase, and β-galactosidase) that possesses a high turnover number. This enzyme reacts with a colorless chromogenic substrate or a chemiluminescent/fluorescent substrate, to generate a reaction product. ELISA assays approach the sensitivity of Radioimmunoassays (RIAs) and have the advantage of being safer and less costly, since they do not depend on radioactive labels. A number of variations of ELISAs have been developed, allowing qualitative detection or quantitative measurement of either antigen or antibody. Most ELISAs are used to measure quantitatively antibody or antigen levels by using a standard curve.


ELISA - General Tips

Read and follow the product instructions. The information contained in the kit manual is your first source of knowledge for your particular assay. If there is any special handling information that you need to be aware of, it will be recorded in the kit insert.
Check the calibration of the pipets that will be used for the assay. 
Do not mix components from different lots or kits. Each component is lot specific and designed to give you a defined level of performance when used before the expiration date.
Always store the kit components at the recommended conditions. 
Do not use the kit past the expiration date marked on the product box. While many of the individual components may have expiration dates that extend beyond the kit date, the total kit expiration date has been determined based on the stability of all the materials stored at the suggested conditions.
Allow the plate to warm to room temperature before opening. The wells in the microtiter plate are coated with an antibody solution. While this coating is stable and robust, moisture will corrupt it causing a decrease in performance. 
Allow all reagents to warm to room temperature before opening unless instructed otherwise. Many of the reagents contain temperature-dependent components that may come out of solution when cold. Using the reagents at room temperature ensures that you have a consistent formulation from assay to assay.
Pre-rinse pipet tips with the reagent before transferring a volume. You need to pre-rinse a tip only once if you are using it to make multiple transfers. Always change the pipet tip if it has been accidentally contaminated.
Always use a fresh pipet tip for each standard, sample or reagent. Do not use the same tip for your standards even when pipetting from the lowest to highest concentrations. 
Pipet the first reagent into the bottom of each well. Pipet subsequent volumes in different locations on the sides of each well, being careful to avoid cross-contaminating reagents
Be careful handling the microtiter wells and reagents to prevent contamination.
Always try to dilute the standards in a matrix as similar to the samples as possible. If the samples are significantly diluted in the assay buffer, then the standards can be diluted in the same buffer as well. If culture media (CM) samples are being analyzed, then the standards should be diluted in non-conditioned media.
Run duplicates for repeatability verification and maximizing your results.


ELISA - Troubleshooting

Weak Color Development

Was the substrate, antibody or conjugate added at the correct point in the assay? 
See the assay procedure provided in the instruction manual.

How long was the substrate incubation? 
It is possible that Stop Solution was added to the plate without allowing the full substrate incubation. 

Were reagents brought to room temperature prior to use? 
It is important to ensure that all reagents are brought to room temperature prior to use, or as mentioned in the product specific instruction manual.

Was the plate read at the correct wavelength? 
See the assay procedure provided in the instruction manual to ensure you are reading the plate at the correct wavelength.

Were the proper volumes of reagents added? 
See the assay procedure provided in the instruction manual. 

How long after the addition of Stop Solution was the plate read?
The plate needs to be read at the correct wavelength as soon as possible after the addition of the Stop Solution.

High Background

How was the plate washed? 
It is important that the plate is washed thoroughly. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem.

What were the incubation times and temperatures? 
If the plate was incubated for too long or at a higher than recommended temperature, high background could result.

Poor Precision

Were the wells washed properly?
All wells receive the same treatment during the wash step. If some are washed less than others, this can translate to poor precision. It is important that the plate is washed thoroughly. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem.

Were the wells aspirated sufficiently after the wash steps?
It is very important that as little Wash Buffer as possible remains in the wells after aspiration. Residual buffer can cause dilution of subsequent reagents.

How were reagents pipetted into wells?
In order to eliminate precision error, remember to pre-rinse all pipet tips used in the assay.

Poor Standard Curve

What was used as the standard diluent?
Diluents other than the supplied assay buffer may contain interfering substances that can affect the standard curve. 

How was the precision of the standard curve? 
If the %CV values for the standard curve signal values (e.g. optical density) are consistently above 5%, it may be a good idea to pay particular attention to the pipetting technique. If the standard curve signal values were acceptable but the sample precision was not, the problem relates to the sample.

How were the standard dilutions prepared?
It is important that test tubes of an appropriate size and material are used. Standard dilutions must be properly mixed (e.g. vortexed) while preparing the serial dilutions.


ELISA - References

The ELISA guidebook (2nd Edition): J.R. Crowther; Methods Mol. Biol. 516, 1-413 (2000)


ELISA - Online Resources

An animated tutorial comparing the direct and indirect ELISA methods


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